The long-term goal of the proposed studies is to determine the molecular mechanism by which streptokinase (SK) activates human fibrinolysis. Understanding this mechanism is significant because it is the basis for the use of SK as a thrombolytic drug for treatment of myocardial infarction. We hypothesize a unified mechanism for the SK-initiated coupled pathway of conformational and proteolytic activation of plasminogen (Pg) to plasmin (Pm). Conformational activation of Pg occurs in a rapid and reversible mechanism accompanied by insertion of IIe1 of SK into the N-terminal binding cleft of Pg, which induces activation of the catalytic site and forms SK.Pg*. The first cycle of Pm formation is initiated by exosite-mediated Pg substrate binding to SK.Pg* and intermolecular cleavage to Pm. The initial cycle acts as a self-limiting triggering mechanism to produce one SK-equivalent of Pm which sequesters SK in the tightly bound SK.Pm complex that in the second cycle catalyzes full conversion of Pg to Pm. The coupled pathway of Pg activation is controlled by intrinsic differences between the affinities of SK for [Glu]Pg, [Lys]Pg, and [Lys]Pm, the equilibrium of [Glu]Pg between compact and extended conformations, and lysine-binding site interactions that enhance SK.Pg* and SK.Pm catalytic complex formation and subsequent Pg substrate recognition. Pg activation by SK is regulated by fibrinogen (Fbg)- and fibrin (Fbn)-promoted assembly of productive complexes through mechanisms that are crucial to the therapeutic activity of SK. Hypotheses addressing major gaps in the understanding of the mechanism of Pg activation by SK and its regulation will be evaluated in equilibrium binding studies employing active site-labeled fluorescent Pg and Pm analogs, steady-state and rapid-reaction kinetics, mutagenesis, and protein structural studies. Specific aims are: (1) To evaluate the hypothesized mechanism of the coupled Pg activation pathway, and to define the roles of Pg conformation and exosite-mediated Pg substrate recognition. (2) To delineate the sequence of molecular events in the mechanism of Pg conformational activation using rapid-reaction kinetics. (3) To determine the roles of SK domains in the mechanism, and to identify Lys residues and pseudo-Lys structures of SK that enhance Pg conformational activation and substrate recognition. (4) To elucidate the mechanism of Fbg and Fbn regulation of SK-initiated fibrinolysis. New information about the SK mechanism and its regulation may enable more effective thrombolytic drugs to be designed.